The present invention relates to peptides derived from maurocalcine capable of penetrating into cells and transporting molecules of interest into these cells.
The problem of the transport of substances, in particular of macromolecules with pharmacological properties, through the plasma membrane and of the access thereof to the various intracellular compartments, in particular the cytoplasmic and nuclear compartments, is an obstacle for biotechnological and biomedical research, and for the pharmaceutical industry.
Among the means currently known for introducing the substances into cells, translocation peptides, also known as CPPs (Cell-Penetrating Peptides) represent particularly advantageous vectors (for a review see, in particular, Prochiantz, Curr. Opin. Cell. Biol., 2000, 12, 400-406; Lindgren et al., Trends Pharmacol. Sci., 2000, 21, 99-102).
Indeed, these small molecules are capable of crossing cell membranes in a transporter- and receptor-independent manner and of transporting macromolecules to which said membranes are impermeable, such as proteins and nucleic acids, at low concentration, without energy, efficiently (transduction of 100% of the cells) and rapidly (of the order of 5 to 15 min), in all cell types, in vivo and in vitro. In addition, it has been shown that some of these peptides are capable of crossing the blood-meningeal barrier (Schwarze and Dowdy, Science, 1999, 285, 1569-1572).
These vectors, which consist of a peptide capable of crossing membranes in a transporter- and receptor-independent manner, are different than other vectors comprising a glycopeptide or a peptide coupled to PEG, in which the positively charged peptide serves to condense the DNA and the PEG or the sugar allows targeting of the cells of interest, in particular by binding of the DNA/glycopeptide complex to the receptor for mannose or asialoglycoprotein (peptides CWCK15CK, CW(CK3)4CK and CWK5CK5CK5C (SEQ ID Nos. 26 to 28): Park et al., Bioconjugate Chem., 2002, 13, 232-239; Kwok et al., J. Pharm. Sciences, 2003, 92, 1174-1185).
The penetrating peptides currently known are divided up into two categories:                peptides derived from membrane translocation signal sequences of various proteins (Kaposi's sarcoma-derived fibroblast growth factor (K-FGF) and immunoglobulin light chain (Ig(v)); the mechanism of penetration of these peptides is unknown, the central hydrophobic region is involved in penetration but the structure of this region varies according to the proteins (alpha-helix (K-FGF) or beta-sheet (Ig(v));        peptides derived from intercellular signaling proteins or “messenger proteins”; these proteins have the particularity of penetrating directly into cells and of reaching the nucleus, where they regulate transcription (HIV-1 Tat, HSV-1 VP22 and homeoproteins).        
Functional studies have made it possible to identify minimum sequences required and sufficient for the translocation of each of these peptides:                the smallest homeoprotein fragment capable of crossing membranes and of serving as a vector to other proteins or to oligonucleotides is the 43-58 peptide, known as penetratin, corresponding to helix 3 of the homeodomain (Derossi et al., J. Biol. Chem., 1994, 269, 10444-10450 and international application WO 97/12912). The study of mutants of this sequence has shown that the alpha-helix structure is not involved in intracellular translocation, but plays a role in nuclear addressing. On the other hand, the W residue at position 48 is important and the amphiphilic properties of the peptide are necessary but not sufficient for the translocation. Supplementary studies have confirmed the role of the W48 residue and shown the importance of the interaction of positively charged amino acids (lysines and arginines) with the membrane phospholipids, which are negatively charged. These studies have led to the inverse micelle model being proposed. According to this model, penetratins are stabilized at the cell surface by electrostatic interactions and the tryptophan at position 48 forces the formation of an inverse micelle which traps the peptide and delivers it into the cytoplasm;        the 267-300 fragment of the VP22 protein corresponds to the minimum sequence for internalization (Elliot and O'Hare, Cell, 1997, 88, 223-233);        the most effective fragment of the Tat protein is the 48-60 fragment, which corresponds to the entire basic region and includes the nuclear localization signal. However, a shorter fragment (47-57) is capable of transporting, in the form of a fusion protein, proteins of 15 to 120 kDa, in various cell types, in vitro and in vivo, and is capable of crossing the blood-meningeal barrier. In addition, unlike the Tat peptide of the human virus, the Tat peptide of the equine virus has a structure similar to that of a homeodomain.        
These functional studies have not made it possible to identify a general mechanism of penetration of these peptides, which would make it possible in particular to identify the common sequence and/or structural elements responsible for the translocation of these peptides.
Maurocalcine (MCa) is a 33 amino acid toxin isolated from the venom of the scorpion Scorpio maurus palmatus, corresponding to the sequence SEQ ID No. 1 in the sequence listing attached in the annex. The corresponding cDNA encodes a 66 amino acid precursor comprising 3 domains: an N-terminal signal peptide of 22 amino acids, followed by a propeptide of 11 amino acids, which is rich in negatively charged amino acids and ends with a cleavage signal characteristic of prohormones (KR), and a C-terminal peptide of 33 amino acids corresponding to the mature peptide (maurocalcine). Maurocalcine exhibits strong homology with the toxin of two other scorpions: imperatoxin of Pandinus imperator (IpTx A, SEQ ID No. 9; 82% identity) and opicalcines 1 and 2 of Opistophthalmus carinatis (SEQ ID Nos. 10 and 11; 91% and 88% identity, respectively; FIG. 1A).
It also exhibits homology, over a 6 amino acid motif comprising a succession of basic residues, followed by a serine or by a threonine (K19K20-K22R23R24-T26), with the activator domain of the II-III loop of the dihydropyridine receptor (DHPR), an L-type voltage-dependent calcium channel. In skeletal muscle, the dihydropyridine receptor—located on the plasma membrane—and the ryanodine receptor type 1 (RyR1)—located in the vesicles of the sarcoplasmic reticulum—form part of the calcium mobilization complex, which is involved in excitation-contraction coupling. Maurocalcine is one of the most potent effectors of the ryanodine receptor type 1 (RyR1); it has in particular been shown that it stimulates the binding of ryanodine to the RyR1 receptor, that it induces considerable modifications in the opening of the calcium channel, characterized by the appearance of prolonged periods of subconductance, and that the extracellular addition of maurocalcine to myotube cultures induces calcium release from the sarcoplasmic reticulum to the cytoplasm (Fajloun et al., FEBS Letters, 2000, 469, 179-185; Estève et al., J. Biol. Chem., 2003, 278, 37822-37831). Thus it has been proposed to use maurocalcine or analogs thereof comprising the KKCKKR motif, as an active ingredient for inducing immunosuppression or treating pathologies related to a calcium channel dysfunction (PCT international application WO 01/64724).
The three-dimensional structure of maurocalcine corresponds to folding according to the ICK motif (Inhibitor Knot Motif), present in many plant, animal or fungal peptides; the ICK family encompasses peptides of different sequences and of varied biological activities, such as animal toxins (snake or spider venom) and protease inhibitors of plant origin, for instance the McoT I-II peptide (SGSDGGVCPKILKKCRRDSDCPGACICRGNGYCG (SEQ ID No. 29)) (Zhu et al., The Faseb Journal, 3 Jul. 2003; Heitz et al., Biochemistry, 2001, 40, 7973-7983).
The structure of maurocalcine consists more specifically of: (i) a compact core which is linked by disulfide bridges (C3-C17, C10-C21 and C16-C32) and which includes three beta-sheets (9-11, 20-33 and 30-33; the sheets 20-33 and 30-33 being anti-parallel), and (ii) an emerging loop at the N-terminal end (Mobash et al., Proteins, 2000, 40, 436-442). It is represented as a molecule comprising a positively charged face which could represent a surface of interaction with the RyR1 receptor (Mobash et al., mentioned above). In addition, the study of maurocalcine mutants (K8A, K19A, K20A, K22A, R23A, R24A and T26A; SEQ ID Nos. 2 to 8) has shown that the R24 residue is important for the effects of maurocalcine on the binding of ryanodine to the RyR1 receptor (Estève et al., mentioned above).
During the course of their study of the effector role of maurocalcine on the ryanodine receptor type 1 (RyR1), the inventors have shown that maurocalcine, which does not belong to any of the categories of proteins containing CPPs mentioned above, is capable of penetrating into cells in vitro and of transporting a protein.
The inventors have now sought to define the minimal characteristics of the amino acid sequences derived from maurocalcine that are capable of serving as a vector for the internalization and addressing of substances of interest, in particular macromolecules of interest such as proteins and nucleic acids, and particles comprising chemical molecules of interest. In addition, since maurocalcine is a toxin with known pharmacological properties, it cannot be used in vivo. Consequently, the inventors have also given themselves the aim of obtaining maurocalcine-derived peptide vectors which are preferably not toxic in vivo, i.e. which do not have a pharmacological activity on the RyR1 receptor, in particular due to the fact that they do not bind to said RyR1 receptor.